1. Field of the Invention
The present invention is directed to a facile synthesis of polyethylene glycol (PEG)-like compounds of defined lengths (i.e., fixed monomer units). Specifically, a method is enumerated for the facile and cost-efficient synthesis of a suitably protected PEG-like spacer, for use under both solid-phase and solution-phase synthesis. More particularly, this invention is directed to a synthetic reaction to produce derivatives of [2-(2-aminoethoxy)ethoxy] acetic acid (AEEA), including the derivative allyloxycarbonyl-[2-(2-aminoethoxy)ethoxy] acetic acid (Alloc-AEEA). This invention is also directed to the use of AEEA derivatives to produce polystyrene-polyethylene-glycol-like (PPL) resins.
2. Description of Related Art
Polyethylene glycols are long chain organic polymers that are flexible, hydrophilic, enzymatically stable, and biologically inert. PEG chains, consisting of the common repeating ethylene glycol entity [—CH2—CH2—O—]n, can be broadly divided into two types: 1) Polymeric PEG-based chains with molecular weights ranging from 1000 to >20,000 and 2) PEG-like chains of molecular weight <1000.
Polymeric PEG-based chains have been used in bioconjugates, and numerous reviews have described the attachment of this linker moiety to various molecules. The popularity of PEG-based technology is evident by the coining of the word “PEGnology,” and by the ready and commercial availability of numerous PEG-based compounds.
As early as 1975, it was shown that PEG chains could help reduce the antigenicity and immunogenicity of proteins. This led to the attachment of PEG chains to various ligands and proteins for use in the fields of biochemistry and medicine. More recently, the hydrophilic character of PEG chains has been utilized in the design of prodrugs. PEG-based chains have also been used as spacers to enhance the fluorescent marker properties of fluorescent biotins. PEGylated DNA adducts have been used to study gene delivery. The amphiphilic nature of the PEG chains have also been utilized extensively to prepare hydrophilic polystyrene (PS)-PEG resins for use in solid-phase peptide synthesis (SPPS) as well as solid-phase organic synthesis (SPOS).
Although PEGylated molecules have numerous advantages as exemplified above, there are also disadvantages associated with these polymeric compounds. The main problem associated with PEG chains has been the lack of well-defined fixed molecular weight of the PEG chains. The variable chain lengths of high molecular weight PEGs (MW=1000 to 20,000 Da) not only impedes purification by size exclusion chromatography and characterization by mass spectrometry, but the problem multiplies if more than one PEG chain is attached per molecule. Thus, advances in analytical chemistry have made the use of polymeric PEG chains impractical in many instances. However, the properties of the PEG-based chains could be mimicked by shorter PEG-like spacers. PEG-like chains exhibit all of the properties of the polymeric PEG chains, but unlike the polymeric PEG chains, PEG-like spacers are made of defined lengths and molecular weights that can be easily controlled. Thus there is a growing technological need for improved PEG-like compounds as opposed to the traditional polymeric PEG chains.
Smaller PEG-like chains made up of between 2 to 6 ethylene glycol units have been used in many applications, especially in cases where the linker properties of the chains are more important than the polymer properties. The short PEG-like linkers can be classified into two types, the homo-[X—(CH2—CH2—O)n]—X and heterobifunctional [X—(CH2—CH2—O)n]—Y spacers. The heterobifunctional PEG-like spacers are becoming more popular mainly due to some recent reports of their synthesis (via multi-step synthetic routes) and applications of such compounds under both solution and solid-phase conditions.
PEG-like chains have primarily been used as spacers and linkers. For example, the homobifunctional PEG-like spacers have been used in the study of bivalent opioid ligands. Bivalent molecules of the type P-X-P, where P represents the pharmacophoric element (β-naltrexamine) and X the ethylene oxide spacer, have been synthesized and tested. It was found that differences in the spacer length (X) led to differences in selectivity of the bivalent ligands towards μ, κ and δ opioid receptors. In another example the commercially available homobifunctional linker, 4,7,10-trioxa-1,13-tridecanediamine was attached to biotin in order to increase its water solubility and to study the stability of this compound towards the enzyme biotindase.
Surprisingly, so far there have been only scant reports in the literature for the synthesis of heterobifunctional spacers. The synthesis of the unprotected diethylene glycol spacer H2N(CH2CH2O)2CH2COOH was reported in 1981, but the synthesis of the protected diethylene glycol spacer wasn't reported until 1995. The diethylene glycol spacer was synthesized independently by two groups and introduced into the peptide chain of calcitonin gene-related peptide (CGRP) and into analogs of atrial natriuretic factor (ANF).
The synthesis of a triethylene glycol spacer, Fmoc-NH(CH2CH2O)3CH2COOR was also reported in 1997 for incorporation into analogues of atrial natriuretic factor (ANF). The structure of Fmoc-AEEA is: 
Recently, the synthesis of the extended tetraethylene glycol spacer units Fmoc-NHCH2CH2COO(CH2CH2O)4X (Fmoc-Ats where X═COCH2CH2COOH, Fmoc-Atg where X═CONHCH2COOH, and Fmoc-Ata where X═CONHCH2CH2COOH) was reported and solid-phase Fmoc/t-Bu based strategy was used for incorporating these spacers into peptides. The interesting spacer BrCH2CONH(CH2CH2O)3CH2COOH was designed and synthesized starting from tetraethylene glycol, and the diethylene glycol spacer maleyl-CH2(CH2OCH2)2COOH starting from H2NCH2(CH2OCH2)COOH. These compounds have been used to crosslink peptides to liposomes via solution chemistry in order to improve the immunogenic response of the small synthetic peptides for use in the development of vaccines for infectious diseases and cancer.
Although there has been a recent spurt in interest in these short chain PEG-like molecules, a low-cost commercial source of these compounds is still lacking. In particular, there is a need or desire for a solid-phase synthesis of labeled peptides (e.g. enkephalin derivatives) containing PEG-like spacers that not only decrease the hydrophobicity of the labeled peptides but also provide easy modulation of the spacer length to ensure accessibility of the labeled peptide to the receptor. Fluorenylmethoxycarbonyl-8-amino-3,6-dioxaoctanoic acid (Fmoc-NH(CH2CH2O)2CH2COOH) is available commercially, and provides the flexibility needed in terms of modulating both the hydrophobicity and spacer length. This spacer unit can be attached to peptides under solid-phase reaction conditions using a commercial reagent, but the high cost of the reagent (U.S.$466/g, Applied Biosystems, Foster City, Calif., U.S.A., and, U.S.$266/g Neosystem Groupe SNPE, Princeton, N.J., U.S.A.) limits the use of the commercial reagent under solid-phase conditions where excess reagent is typically used to drive reactions to completion.
Despite the cost, the prior art synthesis of [2-(2-aminoethoxy)ethoxy)] acetic acid (AEEA) involves four steps starting from commercially available 2-[2-(2-chloroethoxy)ethoxy]-ethanol 2 (Aldrich Chemical Co. Milwaukee, Wis., U.S.A.), as illustrated in Scheme 1. The chloride is first converted into an iodide by heating under reflux with sodium iodide in 2-butanone. The iodide is then converted into a phthalimido derivative 3 by treating it with potassium phthalimide. Oxidation of this compound with Jones reagent leads to the formation of carboxylic acid. Removal of the phthalimido group can be accomplished using hydrazine hydrate to obtain an amine hydrochloride 4. The overall yield after carrying out the four steps was found to be 23%. Although the conversion of the amine hydrochloride to the Fmoc-derivative 1 has not been reported, this compound should be readily available by reaction of AEEA with either Fmoc-Cl or Fmoc-OSu. 
N-terminal analogs of calcitonin gene-related peptide (CGRP) have been synthesized where an AEEA unit was incorporated in the α-helical region of CGRP. The prior art synthesis of N-Fmoc-AEEA starting from 2-(2-aminoethoxy)ethanol is shown in Scheme 2. As shown, 2-(2-aminoethoxy)ethanol is dibenzylated followed by alkylation of the hydroxyl group with sodium hydride and methyl bromoacetate to obtain the methyl ester, which is then hydrolyzed to give the acid. Removal of the benzyl groups then gives AEEA as a white solid. The free amino acid is not isolated, but is converted directly into the Fmoc derivative using Fmoc-Cl. The overall yield of the final product after five steps is approximately 32%. 
The two main drawbacks of the above two schemes are the low overall yields (23% and 32%, respectively) and the necessity for purification (by flash column chromatography, ion-exchange chromatography, etc.) of the intermediates at almost every step. Thus, neither of the two methods is well suited for a low-cost, multi-gram synthesis of the product.
As mentioned, PS-PEG resins are often used in solid-phase peptide synthesis (SPPS) as well as solid-phase organic synthesis (SPOS). Currently there is considerable interest in using solid-phase synthetic methods for the simultaneous preparation of large numbers and quantities of compounds. In the past, solid-phase synthesis was primarily associated with peptide synthesis. However, the current focus for a majority of researchers in the field of solid-phase synthesis is the generation of small drug-like organic molecules, either to generate a new lead or to optimize a known active structure to improve pharmacological and/or pharmacokinetic properties (for example, solubility or in vivo permeability).
PS-PEG resins have been developed that are compatible with a wide array of transformations. PEG-based resins are either composed exclusively of PEG or of PEG supported on a polystyrene or polyamide backbone. Polystyrene has been modified by grafting PEG to the hydrophobic core of PS to produce a polymer that swells in both nonpolar and polar solvents, and thus a broad range of solvents, including water, can be used during synthesis without drastic changes in bed volumes. Modern co-polymers consist of about 60-70% PEG with substitutions in the range of 0.1-0.4 mmol/g. PS-PEG resins exhibit improved physical and mechanical properties and can be used for both batchwise and continuous-flow solid phase synthesis. The excellent coupling and deblocking efficiencies during peptide synthesis on PS-PEG based resins have been attributed to the enhanced solvation of the derivatized PEG. These resins were therefore ideal candidates to be developed for SPOS, however the low initial loading of the reacting functional group (the free amine in the case of an amino resin) on these resins (typically 0.1-0.4 mmol/g) results in small quantities (typically ˜50-100 mg/g of resin) of the molecules being synthesized.
More recently, solid-phase scavengers have been employed in parallel solution phase synthesis in order to purify compounds. Thus, automated parallel purification via nucleophilic and electrophilic scavenging of the resulting byproducts is possible in a cost-effective way using scavenger resins.
PS-PEG graft copolymer resins are prepared by one of two basic methods: (a) by anionic polymerization of ethylene oxide on to the resin to produce the graft resin in situ, e.g. TentaGel™, ArgoGel®, and NovaSyn® resins; or (b) by attachment of preformed PEGs (molecular weight up to ˜3000 Da) to the resin, e.g. PEG-PS (Applied Biosystems) or Novagel® resins.
TentaGel™ resin (RAPP Polymere GmBh, Germany) has been widely used because of its mechanical stability and good swelling properties in organic and aqueous media. It is prepared by grafting ethylene oxide to hydroxymethyl polystyrene by anionic polymerization to give a support with 50-70% PEG content and average graft length of 68 ethyleneglycol units (3000 Da) with typical functional group loading in the range of 0.25-0.3 mmol/g. However, the acid lability of its benzylic ether linkage can be problematic. 
ArgoGel® resin (Argonaut Technologies, San Carlos, Calif., U.S.A.) displays characteristics similar to the TentaGel™ resin. Bifurcation of the graft-polystyrene allows slightly higher loading and greater stability than analogous resins with a benzyl ether linkage. Its PEG content (about 67-82%) and average graft lengths (29-58 repeat units) were optimized to obtain functional group loading in the range of 0.4-0.5 mmol/g. 
The NovaSyn® TG resin (Novabiochem, San Diego, Calif., U.S.A.) overcomes the acid instability problem of TentaGel™ resin by polymerizing ethylene oxide on to a hydroxyethyl polystyrene resin. It is composed of low-cross-linked polystyrene grafted with PEG chains of molecular weight of 3000-4000 terminally functionalized with amino groups. Typical functional loading of the NovaSyn® resin is in the range of 0.2-0.5 mmol/g. 
While the PEG chain is polymerized onto the polystyrene core of TentaGel™, ArgoGel®, and NovaSyn® resins, an alternative form, marketed as PEG-PS resin by Applied Biosystems (Foster City, Calif., U.S.A.), has the preformed PEG chains attached to the polystyrene core via amide bonds. The low-load variety of the resin is prepared by coupling norleucine (as an internal reference amino acid) to functionalized 4-methylbenzhydrylamine (MBHA) polymer. Then a homobifunctional PEG-acid, prepared by reacting the diamino-PEG (molecular weight 2000) with succinic anhydride, is attached to the MBHA-Nle resin, providing the pendant carboxylic acid groups that are finally converted to amino groups (final loading 0.15-0.25 mmol/g) by reacting with ethylenediamine. A modest level of cross-linking also results. 
A “high-load” (˜0.25-0.45 mmol/g) variety of PEG-PS resin was prepared by following a similar strategy except that an ornithine residue [using Fmoc-Orn(Boc)-OH] was inserted instead of norleucine. The Nδ-Boc was removed and a portion of the free amine (25-35%) was capped with acetic anhydride whereas the other half was available for subsequent synthesis. Final loading is typically around 0.6 mmol/g. A percent PEG content of between 40-70% was obtained in both the low-load variety and the high-load variety of PEG-PS, depending on the molecular weight of the diamino-PEG (PEG-600, PEG-900 and PEG-2001). 
In the NovaGel® resin an aminomethylated resin is partially derivatized with methyl-PEG2000-p-nitro-phenylcarbonate. This produces a resin containing approximately 48% PEG, with a substitution of 0.7 mmol/g and good swelling characteristics. Also, the urethane linkage between the core resin and PEG is more stable to both piperidine and TFA (used for deprotection of Fmoc and Boc amine protecting groups, respectively), thus minimizing loss of the PEG chains during synthesis. 
New solid-supports are constantly being developed in order to a) improve the chemical properties of the resin for improved synthesis, b) optimize the physical properties of the beads for better performance and consistency, and/or c) to improve loading capacities of the beads to increase yields. One of the most important parameters that must be considered in designing solid supports is the swelling in various solvents. It is well known that resin beads must be well permeated by both solvents and reagents for the successful completion of any synthesis. Reactions will go to completion only if they are carried out in solvents that adequately swell the resins, and many poor synthetic results are probably due to poor swelling of the resin. For example, dichloromethane (DCM), which hydrogen bonds with the π electrons of the aromatic nuclei of polystyrene, is an excellent swelling solvent for this resin, and therefore syntheses carried out on PS resins in DCM will often go to completion with minimal impurities or side products.
The PEG portion of the PS-PEG resins influences both swelling in polar solvents as well as loading of the functional groups. PS-PEG resins exhibit excellent swelling over a wide range of solvents, from toluene (hydrophobic) to water (hydrophilic), a property that can contribute to a gain in synthetic efficiency. However, introduction of large PEG-based chains decreases resin loading so that loadings are typically much lower for PS-PEG resins (<0.2-0.4 mmol/g) than PS resins (>0.8-2.0 mmol/g). Also, the polymeric nature of the PEG chains can result in variable PEG content of the resin, which in turn affects loading. Thus, there is an interest in an easy and efficient method to obtain PS-PEG like resins with consistently high loading which could be efficiently utilized for both SPPS and SPOS conditions.
Although the PEG-PS based resins have been ideal for the synthesis of peptides, the low substitution level (0.1-0.7 mmol/g) is a problem. A high load resin with better swelling capacities in both hydrophilic and hydrophobic solvents would be very useful for these syntheses.
There is a need or desire for a synthetic reaction for producing AEEA derivatives that is economical and convenient.
There is a further need or desire for a synthetic reaction scheme for producing AEEA derivatives that does not require isolation and purification of intermediates.
There is yet a further need or desire for a synthetic reaction for producing high load resins having the physicochemical properties of PS-PEG resins.